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\begin{document}

\title{Role of C-Jun N terminal kinase (JNK) signaling pathway in acetaminophen
hepatotoxicity}



\author[1]{jijin  zhu}%
\author[2]{WenShang Chen}%
\author[2]{ShiLai Li}%
\affil[1]{Guangxi Medical University First Affiliated Hospital}%
\affil[2]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Acetaminophen (APAP) is a commonly used analgesics and antipyretic
agent. The therapeutic or recommended dose of APAP is not associated
with adverse effects. However, intentional or unintentional overdose of
APAP causes acute liver injury or acute liver failure if treatment is
delayed. Currently, APAP-induced liver injury is one of the major causes
of acute liver injury in the United States and other western countries.
C-Jun N terminal kinase (JNK) implicated in stress-related signaling
pathway plays an indispensable role in the mechanism of APAP
hepatotoxicity. JNK mediates depletion of mitochondrial glutathione in
the metabolic phase and enhances oxidative stress to aggravate liver
injury. In addition, JNK plays an important role in APAP-induced
apoptosis, necrosis or other forms of cell death. Furthermore, JNK plays
a role in regulation of endogenous immune system and aseptic
inflammatory responses induced by APAP. However, JNK may promote cell
regeneration after APAP-induced cell death. The present review therefore
highlights the functions of JNK in APAP-induced liver injury.%
\end{abstract}%



\sloppy


\textbf{Role of C-Jun N terminalkinase (JNK) signaling pathway in
acetaminophen hepatotoxicity}

\textbf{First author: WenShang Chen}

\textbf{Corresponding author: JiJin Zhu}

\textbf{Co-corresponding author: ShiLai Li}

\textbf{Abstract}

Acetaminophen (APAP) is a commonly used analgesics and antipyretic
agent. The therapeutic or recommended dose of APAP is not associated
with adverse effects. However, intentional or unintentional overdose of
APAP causes acute liver injury or acute liver failure if treatment is
delayed. Currently, APAP-induced liver injury is one of the major causes
of acute liver injury in the United States and other western countries.
C-Jun N terminal kinase (JNK) implicated in stress-related signaling
pathway plays an indispensable role in the mechanism of APAP
hepatotoxicity. JNK mediates depletion of mitochondrial glutathione in
the metabolic phase and enhances oxidative stress to aggravate liver
injury. In addition, JNK plays an important role in APAP-induced
apoptosis, necrosis or other forms of cell death. Furthermore, JNK plays
a role in regulation of endogenous immune system and aseptic
inflammatory responses induced by APAP. However, JNK may promote cell
regeneration after APAP-induced cell death. The present review therefore
highlights the functions of JNK in APAP-induced liver injury.

\textbf{Key words} : Acetaminophen; JNK; hepatotoxicity; signaling
pathway;

Acetaminophen (APAP), also known as paracetamol or N-acetyl
para-aminophenol, is a commonly used analgesic and antipyretic agent.
The recommended dose of APAP is usually less than 4g/day and this dose
is considered to be safe {[}1, 2{]}. However, excessive intake of APAP
can cause acute liver injury which may progress to Acute Liver Failure
(ALF). Notably, a previous study reported that a single dose of APAP
\textgreater{} 125mg/kg can cause liver damage {[}3{]}. Currently,
APAP-induced liver injury is a common cause of acute liver injury in the
United States and most western countries.

C-Jun N terminal kinase (JNK) pathway is one of the 3 branches of the
MAPK signaling pathway {[}4{]}. JNK is activated when cells are exposed
to various forms of stress (osmotic stress, oxidative stress and
radiation) or when they are treated with cytokines such as TNF and
IL-1{[}4-6{]}. Therefore, JNK is considered to be a kinase activated by
stress. JNK protein kinases are encoded by three genes, namely; JNK1,
JNK2 and JNK3. JNK1 and JNK2 genes are expressed ubiquitously unlike
JNK3 which has a more limited pattern of expression and is mainly
expressed in the brain, heart and testis{[}4{]}. The aim of the present
review was to explore the role of JNK in APAP-induced liver injury
mainly focusing on JNK1 and JNK2 functions.

Acetyl-L-cysteine (NAC), the only FDA approved remedy for APAP-induced
toxicity is a precursor for GSH that has a therapeutic effect against
APAP overdose. NAC is only effective when administered during early
phases of APAP toxicity, due to its narrow therapeutic window. JNK
signaling pathway plays a significant role in APAP-induced liver injury.
Therefore, JNK is a promising target for treatment of APAP
hepatotoxicity. Therefore, this review summarizes the role of JNK in
APAP-induced toxicity.

\textbf{JNK in Metabolism of APAP in the liver.}

APAP is mainly conjugated with glucuronic acid by enzymes in the
UDP-glucuronosyltransferase (UGT) 1A subfamily and sulfated by
sulfotransferase enzymes at therapeutic doses, to form hydrophilic
compounds in the liver after which are then excreted through bile and
urine. In addition, only a small portion of APAP is metabolized by phase
I enzymes such as cytochrome P450 enzymes, to form the active metabolite
N-acetyl-p-benzoquinone imine (NAPQI). NAPQI is a toxic metabolite that
is detoxified by reduced Glutathione (GSH) to prevent it from binding to
proteins containing sulfhydryl groups, reduce oxidative stress and
prevent liver injury. On the other hand, excess NAPQI generated from an
overdose of APAP results in depletion of GSH in the cytoplasm and
mitochondria {[}7, 8{]}. NAPQI then covalently binds with protein
sulfhydryl groups and forms NAPQI-protein adducts. Previous studies
report that APAP-protein adducts are the main causes of APAP-induced
hepatocyte injury {[}9{]}. However, formation of NAPQI-protein adducts
does not solely cause severe liver injury because 3'-hydroxyacetanilide
(AMAP), a non-toxic regioisomer of APAP in mice, also produces the same
amount of covalent conjugates as APAP {[}10{]}. The difference between
APAP and AMAP is that GSH in the cytoplasm and Endoplasmic Reticulum
(ER) and not the mitochondria, is consumed when AMAP is used. As a
result, there is no activation of various complex pathways associated
with JNK and no liver damage occurs when AMAP is administered{[}11{]}.
Previous studies report that depletion of mitochondrial GSH induces
release of Reactive Oxygen Species (ROS) {[}12, 13{]} and activates the
JNK signaling pathway{[}14{]}. Notably, AMAP is toxic to human liver
cells as it forms mitochondrial protein adducts and causes mitochondrial
dysfunction {[}15, 16{]}. Therefore, formation of NAPQI and protein
adducts may initiate liver injury although they may not be the only
factors implicated in progression of liver injury. Notably, other
downstream events after formation of NAPQI-protein adducts play roles in
APAP-induced liver injury.

The process of liver damage caused by APAP is therefore divided into two
stages;  the metabolism phase which comprises formation of NAPQI,
depletion of GSH and formation of adducts and  the oxidative phase that
comprises oxidative stress, loss of mitochondrial membrane potential and
Mitochondrial Membrane Permeability (MPT){[}17, 18{]}. Notably, APAP or
NAPQI are not required for the oxidative phase and studies report that
adding them into experimental subjects at this phase does not exacerbate
toxicity {[}18{]}. Therefore, oxidative stress amplification through JNK
signaling pathway is the central mechanism in liver damage induced by
APAP.

\emph{In vivo} studies report that depletion of GSH reached a peak after
2 hours of APAP injection, and similar findings are reported for
covalent binding of NAPQI {[}11{]}. However, the metabolism phase is
important because the metabolism of APAP to NAPQI which is mediated by
cytochrome P450 enzymes, is an initial effector of liver injury.
Notably, inhibition of CYP2E1 and CYP1A2 or increase in GSH synthesis
protects the liver against APAP-induced toxicity {[}19{]}. Mitochondrial
bioenergetic is inhibited 2 hours after APAP injection {[}20{]} which
can be attributed to JNK-independent mechanism including depletion of
GSH or covalent binding of APAP{[}21{]}. Moreover, NAPQI-mitochondrial
protein adducts are formed in human hepatocytes and liver
non-parenchymal cells during APAP-induced liver injury {[}22{]}.
Previous studies report that mitochondrial proteins are important
sources of oxidative stress and NAPQI indirectly (by binding with
mitochondrial respiratory compounds or the electron transfer chain) or
directly (by destroying redox cycling through GSH depletion or its redox
reaction) leads to release of ROS {[}23{]}. In addition, a low-dose
(75mg/kg) APAP that dose not form mitochondrial adduct does not cause
JNK activation, therefore no amplification of oxidative stress {[}24{]}.
Furthermore, only the depletion of mitochondrial GSH can induce
sustained JNK activation {[}13{]} and only sustained and not transient
JNK activation is significant. Moreover, transient JNK activation and
reversible mitochondrial dysfunction occur when 150mg/kg APAP is
injected. However, irreversible mitochondrial injury occurs at a dose of
300mg/kg {[}25{]}. Notably, transient activation of JNK does not cause
mitochondrial injury. For instance, a previous study reported that
pretreatment with DAVA (an extract containing Polyphenols) changed
sustained activation of JNK into transient activation, subsequently
improving liver injury {[}26{]}.Therefore, further studies should
explore the effect of mitochondrial GSH depletion and formation of
protein adducts.

Activation of JNK starts 2 hours after injection of APAP and reaches a
peak after 4 hours {[}13{]}. Liver damage gradually occurs after 4-6
hours {[}11{]}. However, the duration of JNK activation is still
controversial. For instance, a previous study reported that JNK
activation starts at about 2-4 hours after APAP injection, reaches a
peak at 6 hours and remains high for 8 hours {[}27{]}. On the contrary a
different study reported that activation of JNK starts 1 hour after APAP
injection {[}28{]}. Moreover, other studies report that activation of
JNK starts 0.5-6 hours after APAP injection {[}26{]}. A previous study
reports that JNK induces changes in the quantity and expression of
glutathione S-transferase A1, to promote liver injury, implying that
activated JNK affects GSH metabolism {[}29{]}. In addition, activation
of JNK mediates formation of peroxynitrite and nitrotyrosine, to induce
oxidative and nitrosative stress {[}29{]}. Therefore, JNK-mitochondrial
signaling loop plays a key role in liver injury.

In summary, JNK signaling pathway is a downstream pathway involved in
depletion of mitochondrial GSH and formation of protein adducts.
Therefore, JNK signaling pathway plays a key role in induction of acute
liver injury by enhancing oxidative stress in APAP-induced liver injury.

\textbf{Activated JNK signaling pathway exacerbates oxidative stress.}

Depletion of GSH and formation of protein adducts in mitochondria
damages the electron transport chain(ETC) participating in ATP
synthesis. The damaged ETC results in production of more ROS{[}30{]}.
Furthermore, depletion of GSH and formation of protein adducts lead to
damage of the mitochondrial antioxidant defense system and inhibits
elimination of ROS {[}30{]}.In addition, NAPQI binds to mitochondrial
respiratory compounds thus inhibiting electron transfer in the
mitochondrial respiratory chain {[}31{]}. Moreover, JNK signaling
cascade is activated to amplify oxidative stress {[}32{]}.

JNK signaling pathway is an important part of the MAPK cascade where MAP
Kinase Kinase Kinase (MAPKKK) is first activated to form activated
ASK-1, MLK3 and GSK3\selectlanguage{english}\selectlanguage{greek}β{[}33-36{]}. This is followed by modification
through phosphorylation thus activating MAP Kinase Kinase (MAPKK) which
ultimately activates JNK {[}37{]}. Phosphorylated JNK and activated
GSK-3\selectlanguage{greek}β \selectlanguage{english}then translocate to the mitochondria {[}34, 36{]}. ASK-1, is
inhibited through binding to thioredoxin in the cytoplasm and
mitochondria {[}38{]}, is activated by dissociating from thioredoxin due
to oxidation of sulfhydryl groups in thioredoxin by ROS {[}39{]}. MLK3
induces JNK phosphorylation within 1 hour of APAP injection {[}28{]}. A
previous study reports that ULK1/2 activates JNK through MKK4/7 and
phosphorylation of Ser403 site of MKK7 by ULK1/2 is necessary for
phosphorylation of Ser271/Thr275 site of MKK7 by MAP3K in APAP-induced
liver injury. Therefore, ULK1/2-dependent phosphorylation of MKK7 is a
crucial step in APAP-induced JNK activation {[}40{]}. Furthermore, MKK4
and MKK7 exhibit a synergistic effect in JNK activation {[}40{]}.
Notably, activity of mTORC1 is inhibited when after administration of an
overdose APAP {[}41{]}, consequently inhibits phosphorylation of ULK1/2.
This enhances activity of ULK1/2 and activation of JNK {[}40{]}. A
previous study divided JNK phosphorylation into the early and late
stages {[}42{]}. The study reported that phosphorylated-GSK-3\selectlanguage{greek}β/\selectlanguage{english}JNK axis
is a major source of APAP-induced liver injury during the early phase
whereas the ASK-1/JNK axis is implicated in liver injury during the late
stages {[}42{]}. Therefore, inhibition of these upstream molecules of
JNK pathway attenuates JNK activation and protects the liver against
APAP-induced toxicity.

Mitochondria act as the upstream organelle for JNK activation and a
downstream target for activated JNK. Therefore, inhibition of JNK
prevents mitochondrial injury in APAP-induced liver injury {[}43{]}.
Moreover, downstream targets for p-JNK are mitochondria under oxidative
stress and not healthy mitochondria {[}11{]}.

In addition, phosphorylated JNK translocates to the mitochondria and
binds to the anchor protein, Sab, located in the outer mitochondrial
membrane {[}44{]}. The effect of p-JNK is gradually amplified after
translocation and binding to Sab. Proteins in the intermembrane space of
mitochondria are released into the cytoplasm following translocation of
p-JNK and binding to Sab. Moreover, protein tyrosine phosphatase
nonreceptor type 6 (SHP1) is released into the cytoplasm from the
mitochondrial Sab channel {[}45{]}. Notably, Sab is required for
sustained activation of JNK and silencing Sab protects against APAP
{[}46{]}. Moreover, JNK-mitochondrial signaling loop is a vicious circle
that continuously amplifies APAP-induced liver damage {[}23{]}.

The main molecular targets for p-JNK are members of the Bcl-2 family.
Translocation of Bax to the mitochondria is a downstream response for
JNK activation after APAP overdose {[}47{]} which has been reported
several models {[}48{]}. P-JNK promotes translocation of Bax to the
mitochondria by directly phosphorylating Bax or by phosphorylating
14-3-3 anchoring Bax in the cytoplasm {[}48, 49{]} thus inducing
mitochondrial Membrane Permeability Transition (MPT). MPT is a common
and important mechanism for different forms of hepatotoxicity. The inner
mitochondrial membrane is permeable to small molecular solutes and the
proton gradient of oxidative phosphorylation is disrupted when the
mitochondria membrane is depolarized {[}50{]}. Loss of mitochondrial
membrane potential results to uncoupling of oxidative phosphorylation
{[}20{]} and swelling of the mitochondria {[}18, 51, 52{]}. Bax is
necessary for MPT {[}2{]} implying that JNK and Bax have a synergistic
effect. A previous study reports that mitochondria are still polarized
3.5 hours post APAP injection but were depolarized after 4.5 hours
{[}47{]}. This observation implies that MPT is a downstream step in JNK
activation. Previous studies divided MPT into two open modes, namely, 
regulatory MPT, which was inhibited by the MPT inhibitor, CsA and
occurred following exposure to a low dose of APAP or a short time and 
non-regulatory MPT, which occurred when a high dose of APAP was
administered {[}53{]}. These modes explain different outcomes from
injection of the above-mentioned high or low doses of APAP, partially or
completely. Release of ROS including superoxide from the mitochondria,
results in oxidation of sulfhydryl groups in MPT pores which is a key
factor for MPT. This is concept was proven through reduction of
disulfides by dithiothreitol reduced thus preventing occurrence of MPT
{[}18, 54{]}. However, studies report contradicting findings on the
roles of Bid and Bcl-xL,the downstream members of Bcl-2 family of p-JNK.
Studies report that the levels of Bax and Bid in the mitochondria
increase after treatment with APAP. However, truncated Bid (tBid) may
not have significant effect on APAP-induced liver injury. In addition,
effect of Bax is blocked by JNK inhibitors although Bid and Bcl-xL are
not affected by these inhibitors {[}11{]}. A different study reports
that Bax and Bid translocate to the mitochondria after an APAP overdose
and inhibition of caspases prevents cleavage of Bid {[}55{]}.
Furthermore, a previous study reports that translocation of Bax to the
mitochondria inactivates Bcl-xL, which is an anti-apoptotic member of
the Bcl-2 family {[}56{]}. Moreover, loss of mitochondrial membrane
potential reduces levels of Bcl-2 {[}31{]}. This implies that
anti-apoptotic members of the Bcl-2 family in the mitochondria are
inhibited despite their role in blocking MPT and levels of members of
Bcl-2 family are affected by APA administration.

Bax, a downstream molecule of JNK activation forms channels by
oligomerization or binding to MPT pores to promote release of cytochrome
C {[}23{]}. Therefore, after the continuously activated JNK translocates
to the mitochondria, mitochondrial respiration is severely inhibited and
production of ROS increases {[}7, 57{]}. Translocation of phosphorylated
JNK and subsequent loss of mitochondrial membrane potential have been
reported in human liver cells {[}58{]} and in a model of UV-induced
oxidative stress {[}59{]}. In addition to cytochrome C, other
intermembrane proteins such as endonuclease G and the Apoptosis-inducing
Factor (AIF) are released which then translocate to the nucleus, leading
to fragmentation of nuclear DNA {[}54, 60{]}. The blockage of release or
translocation to the nucleus of these mitochondrial proteins, downstream
molecules of the p-JNK has protective effect {[}61{]}. Moreover, ROS are
further released thus reacting with the NO produced in this process to
form peroxynitrite and nitrotyrosine. Peroxynitrite is a strong oxidant
that is released into the cytoplasm through passive diffusion or VDAC
anion channel {[}62{]}.

Protein Kinase C\selectlanguage{greek}α\selectlanguage{english}(PKC\selectlanguage{greek}α\selectlanguage{english}) is an important target for p-JNK{[}63{]}. Its
activity increases after APAP overdose and is inhibited by the
conventional JNK inhibitor, SP600125 {[}64{]}. A previous study reports
that after APAP overdose, the levels of PKC-\selectlanguage{greek}α \selectlanguage{english}increase then PKC-\selectlanguage{greek}α \selectlanguage{english}is
translocated to the mitochondria. In the mitochondria it also
phosphorylates mitochondrial proteins and promotes phosphorylation and
translocation of JNK to the mitochondria, subsequently promoting liver
damage {[}63{]}. Moreover, JNK and PKC-\selectlanguage{greek}α \selectlanguage{english}act synergistically to regulate
mitochondrial respiration and mitochondrial-mediated necrotic and
apoptotic cell death {[}21, 65{]}. Therefore, PKC-\selectlanguage{greek}α \selectlanguage{english}and JNK interact
with each other synergistically to participate in feed forward
regulation of APAP-induced liver injury {[}63{]}.

Grb2-associated binder 1 (Gab1) adaptor protein plays an important role
in controlling the balance between death and compensatory proliferation
of hepatocytes during APAP-induced liver injury{[}66{]}. Notably,
silencing Gab1 induces activation of p-JNK and increases translocation
of p-JNK to the mitochondria. Moreover, silencing Gab1 promotes release
of mitochondrial enzymes into the cytoplasm and induces DNA
fragmentation {[}66{]}.

Previous studies report that the Dynamin related protein 1(Drp1), a
downstream molecule of p-JNK, is translocated to the mitochondria, thus
mediating mitochondrial division {[}67, 68{]}. RIPK3, an upstream
molecule of JNK mediates production of ROS in mitochondria {[}69{]}.
Silencing RIPK3 inhibits mitochondrial translocation of Drp1, and
prevents release of mitochondrial AIF and DNA fragmentation {[}67{]}. In
addition, RIPK1 plays a crucial role in sustained activation of
JNK{[}70{]}. Notably, inhibition of RIPK1 prevents translocation of Drp1
to the mitochondria {[}68{]}. Furthermore, inhibition, knockout and/or
silencing of ASK1, MLK3, GSK3b, PKC\selectlanguage{greek}α, \selectlanguage{english}JNK, Sab and cyclophilin D (CypD)
a mitochondrial permeability transition pore regulator{[}71{]}, have
protective effects against APAP {[}68{]}. P-AMPK signaling pathway which
induces autophagy inhibits downstream events of p-JNK although it is
inhibited after APAP administration. Up-regulation of p-AMPK induced by
a PKC inhibitor, exhibits protective effects against APAP toxicity
despite sustained JNK activation {[}63{]}.This is similar to blocking
the release and translocation of endonuclease G and the
Apoptosis-inducing Factor (AIF) to the nucleus (downstream events of
p-JNK), which exhibit a protective effect.

Nrf2 and its Antidote Response Element (ARE) are important antioxidants
in the body. Downstream target genes for Nrf2, including HO-1, NQO1 and
GSH protects the body from oxidative stress {[}72, 73{]}, implying that
Nrf2 alleviates APAP hepatotoxicity. A previous study reports that
upregulation of Nrf2 and its ARE inhibits JNK activation and protects
against APAP-induced oxidative stress {[}74{]}. Furthermore, some
compounds alleviates oxidative stress through AMPK/Akt/Nrf2 in
APAP-induced liver injury {[}31, 75, 76{]}. However, p-JNK can target
Nrf2 to promote its degradation {[}31, 75{]}. Biochemical analysis
showed that p-JNK directly interacts with the Nrf2-ECH homology (Neh) 1
domain of Nrf2 and phosphorylates the serine-aspartate-serine motif 1
(SDS1) region in the Neh6 domain of Nrf2 {[}77{]}. Conversely, a recent
report indicated that the protection of flagellin-induced Nrf2 against
APAP was dependent upon the activation of TLR5-JNK/p38 pathways
{[}78{]}.

In addition, ER stress is an important mechanism for APAP-induced
hepatotoxicity. JNK is an ER stress factor and two key proteins
implicated in ER stress including CHOP and Bim are downstream molecules
of JNK. 4-PBA, an inhibitor of ER stress effectively prevents JNK
activation when administered before APAP injection {[}6{]}. c-Jun, a
p-JNK downstream transcription factor, binds to the 5'regulatory region
of CHOP gene {[}79, 80{]} and mediates transcription of the gene. Parkin
is a post-translational regulation factor of CHOP that induces
degeneration of CHOP through one of the branches of UPR, the
PERK/eIF2\selectlanguage{greek}α/\selectlanguage{english}ATF4 pathway. Notably, c-Jun can competitively bind to the
binding site of ATF4 in the Parkin gene promoter{[}74{]}. Therefore,
JNK/c-Jun can inhibit expression of Parkin {[}81{]}.

Inhibition of JNK-dependent p53 up-regulated Modulator of Apoptosis
(PUMA), a downstream molecule of JNK, significantly improves hepatocyte
necrosis {[}82{]}. NF-\selectlanguage{greek}κ\selectlanguage{english}B response gene products inhibit JNK {[}83{]} but
the process is blocked by depletion of GSH {[}17{]}.

Some studies report that only JNK2 is involved in amplifying the effect
of oxidative stress by translocating to the mitochondria whereas JNK1
has not significant effects on oxidative stress {[}84{]}. However, other
studies report that both JNK1 and JNK2 translocate to the mitochondria,
play a role in amplifying oxidative stress and participate in activation
of downstream events. In addition, JNK1 has a greater effect on
mitochondrial bioenergetics compared with JNK2 {[}11{]}. Therefore, the
specific role of the two regioisomers of JNK should be explored further.

In summary, after depletion of mitochondrial GSH by NAPQI and formation
of covalent adducts with mitochondrial proteins, oxidative stress,
(amplified by the JNK signaling pathway and JNK-related signal
transduction pathways) exerts a ``second hit''{[}26{]} on damage of
liver cells. Therefore, this process is a synergistic event that leads
to decreased mitochondrial respiration and bioenergetics, mitochondrial
dysfunction and eventually severe liver damage.

\textbf{JNK mediates APPA-induced necrosis, apoptosis and other forms of
cell death}

\emph{In vivo} and \emph{in vitro} studies report that depletion of
mitochondrial GSH results in necrosis {[}85, 86{]}. Depletion of
mitochondrial GSH by DEM results in a significant increase in necrosis
{[}4, 13{]}. Notably, DEM depletes cytoplasmic and mitochondrial GSH,
leading to 100\% necrosis instead of apoptosis despite exposure to TNF-\selectlanguage{greek}α\selectlanguage{english}
{[}13{]}. Administration of low concentration of DEM or APAP only
depletes cytoplasmic GSH in hepatocytes, therefore, they become
sensitive to TNF-\selectlanguage{greek}α-\selectlanguage{english}induced apoptosis {[}13, 87{]}. Moreover, using
GSH-EE to restore depleted GSH can reverse sensitivity to apoptosis,
induced by TNF-\selectlanguage{greek}α\selectlanguage{english} {[}87{]}. Furthermore, antioxidants can restore the
levels of GSH and reduce levels of p-JNK {[}88, 89{]}. Previous studies
report that JNK regulates apoptosis and necrosis {[}4, 45, 81{]}.

\textbf{Apoptosis:}

JNK promotes cytokine receptor apoptosis signaling pathway. Previous
studies report that JNK activation promoted the of release of Fas-L in
an autocrine or paracrine manner to increase toxicity in adjacent liver
cells {[}4, 90{]} and induce the cytokine receptor death signaling
pathway. JNK is activated during APAP-induced oxidative stress in
hepatocytes and non-parenchymal cells such as sinusoidal endothelial
cells. On the other hand, inflammatory cells release pro-inflammatory
cytokines such as TNF-\selectlanguage{greek}α \selectlanguage{english}and INF-\selectlanguage{greek}γ, \selectlanguage{english}thus promoting inflammatory reactions
and aggravating liver damage under inflammatory conditions {[}91, 92{]}.
The pro-apoptotic factors, p53 and c-Myc, are phosphorylated by
activated JNK in cells exposed to stress factors {[}4{]}. Therefore,
sustained activation of JNK mediates TNF-\selectlanguage{greek}α\selectlanguage{english} {[}93{]} or FasL-induced
apoptosis. Although endotoxin-induced production of TNF-\selectlanguage{greek}α \selectlanguage{english}does not
increase APAP-induced liver damage {[}94{]}, Astaxanthin (ASX) reduces
apoptosis by inhibiting TNF-\selectlanguage{greek}α-\selectlanguage{english}mediated JNK signaling pathway {[}95{]}.
Furthermore, TNF\selectlanguage{greek}α-\selectlanguage{english}induced apoptosis following pretreatment with APAP, is
inhibited by SNAP (producing NO). Furthermore, SNAP inhibits 50\% of
caspase3 activity, which is increased by combined treatment with APAP
and TNF-\selectlanguage{greek}α\selectlanguage{english} {[}13{]}. In addition, silencing TNF-\selectlanguage{greek}α \selectlanguage{english}or TNFR1 exhibits
protective effects against APAP-induced liver damage {[}96{]}. A
previous study reported that JNK inhibitors partially block
TNF-\selectlanguage{greek}α-\selectlanguage{english}induced apoptosis in cells treated with APAP, without affecting
GSH consumption {[}13{]}. Activation of TNF receptor induces activation
of caspase 8, which in turn truncates Bid into tBid then tBid is
translocated to the mitochondria. Bax is also translocated to the
mitochondria where it forms MPT pores with Bak and Bad. These changes
induce release of cytochrome C and AIF from mitochondria and
translocation to the nucleus. As a result, apoptotic bodies are formed,
caspase9 and caspase3 are activated in presence of sufficient ATP.
Notably, the TNF superfamily activates death receptors including TNF\selectlanguage{greek}α,
\selectlanguage{english}(Fas ligand)FasL and TRAIL. FasL is the most harmful to hepatocytes.
Moreover, sensitivity of cells to FasL and toxicity to adjacent liver
cells increases with release of cell contents {[}97{]}. TRAIL which is a
homologue of TNF-\selectlanguage{greek}α, \selectlanguage{english}induces apoptosis in hepatocytes, a process which is
independent of direct activation of caspases but is dependent on the
activation of JNK and Bim. Therefore, inhibition of JNK and Bim protects
hepatocytes against death induced by FasL or TNF-\selectlanguage{greek}α\selectlanguage{english} {[}97-99{]}.
Furthermore, p-JNK phosphorylates Bim {[}100{]}, Bax and Bak whereas
Bcl-2 and Bcl-xL are inhibited during APAP-induced hepatotoxicity.
Therefore, these findings imply that TRAIL induces apoptosis in
hepatocytes through the JNK-Bim axis. Previous studies report that
extramitochondrial depletion of GSH alters the thiol-disulfide redox
state, leading to inhibition of transactivation of NF-kB and sustained
activation of JNK, which ultimately induce sensitivity to TNF-\selectlanguage{greek}α-\selectlanguage{english}induced
apoptosis {[}13{]}. The JNK pro-apoptosis pathway, P38 and NF-kB
pro-survival signaling pathways are simultaneously activated {[}101{]}.
Sustained activation of JNK plays an essential role in primary
hepatocytes sensitive to TNF\selectlanguage{greek}α-\selectlanguage{english}induced apoptosis by inhibiting NF-kB
{[}93{]}. Therefore, JNK plays an important role in promoting apoptosis
in liver cells, induced by death receptor signaling during APAP
hepatotoxicity. Additionally, sensitivity to TNF\selectlanguage{greek}α-\selectlanguage{english}induced apoptosis in
liver cells treated with APAP may promote APAP-induced liver damage.
This explains why the dose of APAP required to induce acute liver injury
in patients with basic liver diseases such as chronic hepatitis B,
alcoholic liver and non-alcoholic fatty liver, is lower compared with
that of patients without underlying diseases.

JNK plays a role in stress-induced apoptosis through the mitochondrial
pathway {[}4{]}. Defect of JNK-deficient fibroblasts in stress-induced
apoptosis is mainly located in the mitochondria {[}59{]}. Previous
studies report that translocation of p-JNK to the mitochondria induces
release of cytochrome C and SMAC from the mitochondrial intermembrane
space, leading to apoptosis {[}65, 102{]}. Notably, release of
cytochrome C from the mitochondria is inhibited by the absence of JNK
{[}103{]}. Additionally, sustained activation of JNK promotes cell
damage or death {[}93, 104{]}. The potential targets for p-JNK for
regulation of release of cytochrome C are the apoptotic regulatory
members of the Bcl-2 family{[}4{]}. In addition, p-JNK translocation to
the mitochondria promotes translocation of Bax to the mitochondria thus
activating caspase 9, leading to apoptosis through the intrinsic
pathway. Moreover, the anti-apoptotic proteins Bcl-2 and Bcl-xL are
phosphorylated by JNK {[}105, 106{]}. Therefore, apoptosis through the
JNK-dependent but transcription-independent pathway, (the
mitochondrial/caspase9 pathway), is implicated in APAP-induced cell
death.

Notably, only sustained rather than transient activation of JNK is
associated with apoptosis {[}107{]}, which is consistent with the
mechanism of APAP-induced liver injury. For instance, TNF-\selectlanguage{greek}α \selectlanguage{english}causes
transient JNK activation with no apoptotic response {[}13, 108{]}.
Although several studies report that JNK-mediated apoptosis is involved
in APAP-induced liver injury, the relationship between JNK-mediated
apoptosis and APAP-induced liver injury has not been fully explored.
Several studies report that apoptosis is not involved in APAP-induced
liver injury because caspases, especially caspase 3, is not activated
during APAP hepatotoxicity {[}109{]}. Furthermore, a previous study
reported that the levels of activated caspase 3 did not increase in
human hepatocytes after an APAP overdose {[}110{]}. Moreover, previous
studies report that pan-caspase inhibitors do not alleviate APAP-induced
liver damage {[}86, 111{]}. However, TRAIL induces apoptosis in
hepatocytes and in a process that is not dependent on the direct
activation of caspases but dependent on activation of JNK and Bim
{[}97{]}. This observation partially explains why caspase 3 is not
activated during APAP-induced liver injury as reported by some studies.
However, recent studies report that caspase 3 is activated during
APAP-induced liver damage {[}42, 112, 113{]}. In addition, hydrogen
sulfide inhibits apoptosis through the JNK pathway, thus alleviating
APAP-induced liver damage {[}114{]}. Several compounds protects liver
cells against APAP-induced toxicity through anti-apoptotic mechanisms
{[}115, 116{]}. Previous \emph{in vitro} studies report a decrease in
cell viability and a significant increase in levels of apoptotic cells
after APAP administration {[}74{]}. Notably, treatment with caspase 3
inhibitors completely blocks APAP-induced apoptosis {[}47{]}.

\textbf{Necrosis:}

Necrosis is the main form of liver-cell death during APAP-induced liver
injury. Previous studies report that APAP-induced liver injury does not
involve apoptosis {[}117{]}, as mentioned above. Studies report that JNK
inhibitors block APAP-induced necrotic cell death {[}13{]}. Notably,
APAP-induced death of liver cells is not characterized by cell
shrinkage, nuclear pyknosis and other common cytological features of
apoptosis. On the contrary, cytological manifestations APAP-induced
liver damage mainly include extensive mitochondrial dysfunction and
nuclear lysis accompanied by swelling of cells and organelles and
release of cellular contents. These features show that APAP-induced cell
death is a oncotic necrosis process {[}86{]}.Mitochondrial respiration
is inhibited after translocation of p-JNK to the mitochondria and ATP
production is significantly reduced, leading to insufficient energy for
activation of caspase and formation of apoptotic bodies. Previous
studies report that when fructose and glycine are administered along
with APAP, a significant increase in ATP production is observed and,
cells are protected from necrosis although MPT was still normal and an
increase in apoptosis was observed {[}47{]}. In addition, endonuclease G
and the Apoptosis-inducing Factor (AIF) are released from the
mitochondria, translocated to the nucleus, where they induce nuclear DNA
fragmentation after JNK activation and ultimately initiate necrosis in
cells {[}118{]}. In addition, apoptosis requires a reduced environment
for caspase activation and the redox state of liver cells may affect
apoptosis during APAP-induced liver injury {[}11, 119,
120{]}.Furthermore, Damage-associated Molecular Pattern (DAMP) which
induces inflammation, is released and interacts with pattern recognition
receptors including toll-like receptors(TLR) on inflammatory cells.
These events lead to release of pro-inflammatory cytokines (such as
TNF\selectlanguage{greek}α\selectlanguage{english}and INF-\selectlanguage{greek}γ\selectlanguage{english}) and induction of an inflammatory response {[}91, 92{]}.
These are generally the characteristics of necrosis since apoptosis is
not characterized by release of cellular contents. TRAIL-JNK-Bim axis is
important in APAP-induced cell necrosis, and deletion of TRAIL or Bim
protects hepatocytes and sinusoidal endothelial cells from necrosis
{[}100, 121{]}. Therefore, ability of TRAIL-JNK-Bim axis in
amplification of necrosis in liver cells is greater compared with the
ability of the Bcl-2 family members to induce apoptosis through the
mitochondrial pathway {[}97{]}.

Previous studies also report that hepatocytes undergo both necrotic and
apoptotic cell death in APAP-induced hepatotoxicity {[}47, 122{]}. In
addition, upstream events such as JNK activation and translocation to
the mitochondria, induction of pro-apoptotic Bcl-2 homologs and increase
in MPT with subsequent release of cytochrome C and AIF, are indicators
of apoptotic cell death. However, inhibition of mitochondrial
respiration and ATP synthesis cannot solely induce apoptosis. Moreover,
JNK and MPT mediate apoptosis and necrosis. Therefore, apoptosis is the
main form of cell death in the early stages of APAP hepatotoxicity
whereas necrosis mainly occurs in the late stages {[}113, 123{]}. A
previous study reports presence of caspase-cleaved cytokeratin-18 in
patients with an APAP overdose, indicating that apoptosis of hepatocytes
occurred in the early stages of APAP-induced acute liver injury
{[}124{]}.

However, several studies challenged the widely accepted conclusion that
JNK mediates necrotic or apoptotic cell death in APAP-induced liver
injury. For instance, knockout of Gst-pi, a negative regulator of JNK,
protects mice from APAP-induced liver damage {[}125{]}. In addition,
simultaneous activation of JNK1 and JNK2 in mice protected them against
APAP-induced necrotic cell death by regulating oxidative stress
response. Moreover, lack of JunD activation in hepatocytes with
specifical knockout of JNK1 and JNK2 (Jnk[?]hepa), shows that
JNK-JunD-dependent mechanism may be involved in protection of liver
cells against APAP-induced liver damage {[}109{]}. Nevertheless, JNK
signaling pathway plays a central role as it modulates necrotic or
apoptotic cell death in APAP-induced liver injury probably through a
mechanism that accelerates and amplifies oxidative damage.

In summary, JNK-mediated necrotic and apoptotic cell death are involved
in APAP-induced liver injury and the specific form of death may depend
on different conditions.

\textbf{Necroptosis and other Forms of Cell Death}

Several signaling pathways involved in stress initiation, amplification,
expansion and ultimately cell death, have been identified in
APAP-induced liver injury thus promoting the use of the term, programmed
necrosis {[}126{]}. Previous studies report that RIPK1 and RIPK3 combine
and translocate to the mitochondria to mediate necroptosis {[}67,
127{]}. In addition, RIPK3 and MLKL are up-regulated after APAP
injection, in a time and dose-dependent manner similar to JNK {[}128{]}.
Notably, JNK is a biomarker of UPR and ER stress. However, other studies
report that necroptosis does not affect cell death in APAP-induced
toxicity {[}68{]}. Moreover, additional studies report several forms of
cell death during APAP hepatotoxicity including initial necrosis
followed by pyroptosis, apoptosis and necroptosis {[}129{]}.

In summary, JNK-mediated death of liver cells is necessary for
APAP-induced liver toxicity and several protective effects are exhibited
through inhibition of JNK. For instance, the tumor suppressor, P53,
protects liver cells against APAP-induced liver damage by inhibiting JNK
activation {[}130{]}. Moreover, 4MP inhibits JNK activation and p-JNK
translocation to the mitochondria, thus protecting liver cells against
APAP-induced liver damage {[}131, 132{]}. Furthermore, the Gadd45\selectlanguage{greek}β
\selectlanguage{english}agonist inhibits phosphorylation of MKK4 and JNK {[}133{]} whereas
Metformin inhibits JNK thus exhibiting protective effects against APAP
through Gadd45\selectlanguage{greek}β\selectlanguage{english} {[}134, 135{]}. Additionally, quercetin offers
protection against APAP-induced liver injury by reducing JNK activation
{[}136, 137{]}. Inhibition or inactivation of JNK can protect liver
cells against APAP-induced liver injury {[}89, 138, 139{]}.

\textbf{Role of JNK in Liver Endogenous Immunity and Non-parenchymal
Cells}

JNK-mediated hepatocyte necrosis releases DAMP which interacts with
pattern recognition receptors (such as toll-like receptors) on
inflammatory cells, leading to release of pro-inflammatory cytokines
(such as TNF\selectlanguage{greek}α\selectlanguage{english}and INF-\selectlanguage{greek}γ\selectlanguage{english}) thus promoting inflammatory responses. In
addition, a previous study reports that phosphorylation of JNK was
significantly inhibited after silencing TLR4 in the RAW264.7 immune cell
line {[}140{]}. Therefore, TLR4 can activate JNK signaling pathway to
induce an inflammatory response and infiltration of macrophages thus
promoting APAP-induced liver injury {[}140{]}. Also, it has been
reported that TLR3 can act on JNK to be injury-provoking in the model of
APAP-induced liver injury {[}141{]}. Hematoxylin-eosin (HE) staining
shows significant infiltration of inflammatory cells in areas with liver
injury {[}142{]}. Therefore, the endogenous immune system, including
liver macrophages, is induced during APAP hepatotoxicity, causing
aseptic inflammation {[}143{]}. Moreover, previous studies report an
increase in release of inflammatory factors such as TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β \selectlanguage{english}and
IL-6 and immune cells such as neutrophils, macrophages, NK cells and NKT
cells are recruited. These factors may exacerbate liver damage through
the effect of NKT cells and effectors such as INF-\selectlanguage{english}\selectlanguage{greek}γ{[}97{]}.
Additionally, neutrophil-mediated damage, secondary to hepatocyte
necrosis, may promote APAP-induced liver injury {[}144{]}.
Mannan-binding Lectin (MBL), a molecule in the innate immune system that
is mainly produced by the liver, promotes JNK activation and
up-regulates nuclear expression of Specificity Protein 1 (SP1) to
aggravate liver damage {[}145{]}. Previous studies report that JNK
regulates secretion of IL-2, proliferation of CD8+ T cells and
differentiation of CD4+ T cells into helper T cells {[}146{]}.
Furthermore, knockout of TNF or TNFR1 protects liver cells against
APAP-induced damage. These findings show that JNK regulates immune cells
or the immune factor TNF or FasL-induced injury of liver cells during
APAP-induced liver injury. However, these immune or inflammatory cells,
including NK cells and NKT, can also play protective roles
{[}147-149{]}. Furthermore, mice devoid of the interferon Fas or Fas
ligand did not show APAP-induced liver damage whereas those that lacked
interleukin-10 and interleukin-6 were protected against liver damage
{[}150{]}.

Therefore, \emph{in vivo} studies provide reliable findings compared
with \emph{in vitro} experiments on the role of the endogenous immune
system in APAP-induced liver injury (regardless of whether it aggravates
damage or offers protection). In addition, should be carried out to
explore the specific role of JNK in regulation of immune cells in
APAP-induced liver injury.

A previous study reports that JNK1 is highly induced and a significant
increase in levels of p-JNK is observed in mice with specific knockout
of JNK1 and JNK2 in their hepatocytes. In addition, the levels of
infiltration in the liver explains the phenomenon of JNK activation
after APAP administration {[}109{]}. Notably, a previous study using a
hepatocellular carcinoma model reported that the effect of JNK in
promoting development of liver cancer mainly involves non-parenchymal
cells and not hepatocytes {[}151{]}. Furthermore, sinusoidal endothelial
cells are more sensitive compared with hepatocytes to APAP-induced liver
injury {[}152{]}. Therefore, studies on the role of JNK in APAP-induced
liver injury should also focus on the effects on non-parenchymal cells.

\textbf{Role of JNK in Restoration and Regeneration Phase}

Proliferation and recovery of cells are extremely important after
APAP-induced cell death. Inhibition of EGFR in liver cells, 12 hours
after APAP injection, leads to impairment in proliferation, persistence
of liver injury and an increase in mouse mortality {[}153{]}. JNK plays
an important role in cell proliferation {[}59{]}. Therefore, knockout of
JNK1 inhibits progression of liver cancer and proliferation of
hepatocytes is significantly affected. In addition, JNK1 is involved in
transformation of hepatic stellate cells into fibroblasts. Notably,
inhibition of JNK attenuates fibrosis in models of bile duct ligation or
CCl4-induced liver injury {[}154{]}. Therefore, JNK may promote
regeneration of liver cells during the late stages of APAP-induced liver
injury and protect liver cells against APAP hepatotoxicity. Furthermore,
recent studies report that JNK may be critical to cell survival
{[}155{]} because JNK promotes recovery and regeneration of liver cells
after APAP-induced death of liver cells. Additionally, silencing CHOP,
which is a downstream gene of JNK, promotes proliferation of hepatocytes
during APAP-induced liver injury {[}117{]}. However, further studies
should explore the role of JNK in recovery and regeneration of cells
following APAP-induced liver injury.

\textbf{Conclusion}

Intentional or unintentional overdose of acetaminophen leads to
Acetaminophen-induced liver injury, which is the most common cause of
acute liver injury in the United States and most western countries.
C-Jun N terminal kinase (JNK) may is a potential target for treatment of
APAP-induced liver injury, therefore, this review explored the essential
role of JNK signaling pathway in various stages of APAP hepatotoxicity.
JNK signaling pathway mediates APAP metabolism, especially GSH
metabolism, amplifies mitochondrial oxidative stress, accelerates injury
of liver cells, and mediates apoptotic or necrotic and other forms of
cells death. In addition, JNK signaling pathway play a key role in
endogenous immunity and non-parenchymal cells. Moreover, JNK signaling
pathway induces regeneration of liver cells after cell death.

\textbf{Main Concepts}

JNK signaling pathway, a downstream cascade in the depletion of
mitochondrial GSH and formation of protein adducts in mitochondria, is
implicated in GSH metabolism.

JNK signaling pathway, one of the 3 branches of the MAPK signaling
pathway is activated by MAP3K such as ASK-1, MLK3 and GSK3\selectlanguage{greek}β\selectlanguage{english}to MAP2K thus
increasing mitochondrial oxidative stress. Upstream factors of JNK
signaling pathway include ULK1/2, PKC\selectlanguage{greek}α, \selectlanguage{english}Gab1, RIPK1, RIPK3, and the
molecules related in ER stress and UPR.

JNK-mitochondrial signaling loop, a vicious circle, plays a central role
in mechanism of APAP-induced liver injury to amplify oxidative stress.

JNK-mediated necrotic, apoptotic or other forms of cell death are
dependent on different conditions in APAP-induced liver injury.

JNK signaling pathway in Liver Endogenous Immunity and Non-parenchymal
Cells can promote liver cells against injury or may protect liver cells
against APAP hepatotoxicity.

JNK signaling pathway improves restoration and regeneration of liver
cells in the restoration and regeneration phase thus protecting liver
cells.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Figure 1 & JNK signaling pathway during APAP-induced liver injury. An
overdose of APAP produces excessive NAPQI, depletes GSH in the
cytoplasm, endoplasmic reticulum and mitochondria, forms mitochondrial
protein adducts, damages the electron transport chain and induces
initial oxidative stress and production of ROS. In addition, MAP3K,
including MLK3, ASK1 and GSK3\selectlanguage{greek}β \selectlanguage{english}are activated,resulting in JNK
activation through MAP2k. Moreover, mTORC1 is inhibited during
APAP-induced liver injury and thus inhibiting phosphorylation of ULK1/2,
increasing level of activated ULK1/2 which ultimately acts on JNK.
Upstream molecules of JNK include RIPK1 and RIPK3. Endoplasmic Reticulum
(ER) stress is an important mechanism in APAP-induced liver injury and
JNK is a biomarker of ER stress. Notably, phosphorylated JNK
translocates to the mitochondria and binds to the adaptor protein, Sab.
The downstream molecules of p-JNK, such as Bax and Drp1 are also
translocated to the mitochondria. Furthermore, downstream events of
p-JNK include PKC-\selectlanguage{greek}α, \selectlanguage{english}CHOP and Bim. Bax mainly induces mitochondrial
dysfunction, MPT and continuous production of ROS. In addition, ROS
produced in the mitochondria play an essential role in sustaining
activation of JNK. Notably, MPT pores open and endonuclease G,
cytochrome C and AIF are released into the cytoplasm and translocate to
the nucleus. Finally, death or injury of liver cells
occurs.\tabularnewline
Figure 2 & JNK mediates APAP-induced death of liver cells. p-JNK
promotes secretion of FasL in an autocrine or paracrine manner. In
addition, production of TNF-\selectlanguage{greek}α \selectlanguage{english}and TRAIL increases when inflammation
occurs and these molecules are associated with the corresponding death
receptors. Moreover, JNK signaling pathway is activated to amplify
oxidative stress during APAP-induced hepatotoxicity. Thereafter, JNK
mediates death receptor-dependent apoptosis. Additionally, p-JNK
translocates to the mitochondria and activates the downstream apoptotic
events to mediate apoptosis through the mitochondria/caspase-9 pathway.
Furthermore, p-JNK in the mitochondria induces the downstream events in
the mitochondria. MPT pores open, mitochondrial respiration is inhibited
and ATP synthesis is decreased resulting in insufficient energy for
apoptosis. Finally, mitochondrial swelling and oncotic necrosis
occurs.\tabularnewline
\bottomrule
\end{longtable}

\textbf{References:}

{[}1{]}. A. M. Larson, J.P.R.J., Acetaminophen-induced acute liver
failure: results of a United States multicenter, prospective study.
Hepatology, 2005. 42(6): p. 1364-72.

{[}2{]}. Ramachandran, A. and H. Jaeschke, Acetaminophen Hepatotoxicity.
Seminars in Liver Disease, 2019. 39(02): p. 221-234.

{[}3{]}. N. A. Buckley, I.M.W.D., Oral or intravenous N-acetylcysteine:
which is the treatment of choice for acetaminophe(paracetamol)
poisoning?, Journal of toxicology. Clinical toxicology, 1999. 17(6): p.
759-767.

{[}4{]}. Davis, R.J., Signal Transduction by the JNK Group of MAP
Kinases. Cell, 2000. 103(2): p. 239-252.

{[}5{]}. Ip, Y.T. and R.J. Davis, Signal transduction by the c-Jun
N-terminal kinase (JNK)--from inflammation to development. Curr Opin
Cell Biol., 1998. 10(2): p. 205-19.

{[}6{]}. Shimizu, D., et al., Protection afforded by pre- or
post-treatment with 4-phenylbutyrate against liver injury induced by
acetaminophen overdose in mice. Pharmacological Research, 2014. 87: p.
26-41.

{[}7{]}. Jaeschke, H.K.T.R., The role of oxidant stress and reactive
nitrogen species in acetaminophen hepatotoxicity. Toxicol. Lett, 2003.
144: p. 279--288.

{[}8{]}. James LP, M.P.H.J., Acetaminophen-induced hepatotoxicity. Drug
Metab Dispos, 2003. 31: p. 1499 --1506.

{[}9{]}. Amar, P.J.A.S., Acetaminophen safety and hepatotoxicity-where
do we go from here? Expert Opin. Expert Opin Drug Saf, 2007. 6: p.
341--355.

{[}10{]}. Myers, T.G.D.E. and S.D.A.N. Cohen, A comparative study of
mouse liver proteins arylated by reactive metabolites of acetaminophen
and its nonhepatotoxic regioisomer, 3 -hydroxyacetanilide. Chem. Res.
Toxicol., 1995. 8: p. 403--413.

{[}11{]}. Hanawa, N., et al., Role of JNK Translocation to Mitochondria
Leading to Inhibition of Mitochondria Bioenergetics in
Acetaminophen-induced Liver Injury. Journal of Biological Chemistry,
2008. 283(20): p. 13565-13577.

{[}12{]}. Han, D.C.R.R., Effect of glutathione depletion on sites and
topology of superoxide and hydrogen peroxide production in mitochondria.
Mol. Pharmacol., 2003. 64: p. 1136--1144.

{[}13{]}. Matsumaru, K., Mechanisms for sensitization to TNF-induced
apoptosis by acute glutathione depletion in murine hepatocytes.
Hepatology, 2003. 37(6): p. 1425-1434.

{[}14{]}. Nga T Nguyen, K.D.J.Y., Mitochondrial protein adduct and
superoxide generation are prerequisites for early activation of c-jun
N-terminal kinase within the cytosol after an acetaminophen overdose in
mice. Toxicol Lett, 2021. 338: p. 21-31.

{[}15{]}. Xie, Y., et al., Mitochondrial protein adducts formation and
mitochondrial dysfunction during N-acetyl-m-aminophenol (AMAP)-induced
hepatotoxicity in primary human hepatocytes. Toxicology and Applied
Pharmacology, 2015. 289(2): p. 213-222.

{[}16{]}. Cliff Rowe, M.S.E.L., Perfused human hepatocyte microtissues
identify reactive metabolite-forming and mitochondria-perturbing
hepatotoxins. Toxicology in Vitro, 2018. 46: p. 29-38.

{[}17{]}. Xu, J.J., B.S. Hendriks and J. Zhao, Multiple effects of
acetaminophen and p38 inhibitors: Towards pathway toxicology. FEBS
Letters, 2008. 582.

{[}18{]}. Reid, A.B., et al., Mechanisms of Acetaminophen-Induced
Hepatotoxicity: Role of Oxidative Stress and Mitochondrial Permeability
Transition in Freshly Isolated Mouse Hepatocytes. Journal of
Pharmacology and Experimental Therapeutics, 2005. 312(2): p. 509-516.

{[}19{]}. Zaher, H.B.J.T. and S.S.T.C. A. M., Protection against
acetaminophen toxicity in CYP1A2 and CYP2E1 double-null mice. Toxicol.
Appl. Pharmacol. Toxicol. Appl. Pharmacol., 1998. 152: p. 193--199.

{[}20{]}. Donnelly PJ, W.R.R.W., Inhibition of mitochondrial respiration
in vivo is an early event in acetaminophen-induced hepatotoxicity. Arch
Toxicol, 1994. 68(2): p. 110--118.

{[}21{]}. Zhou Q, L.P.H.D., c-Jun N-terminal kinase regulates
mitochondrial bioenergetics by modulating pyruvate dehydrogenase
activity in primary cortical neurons. J Neurochem, 2007. 104(2): p.
325--335.

{[}22{]}. Bruderer, R.B.O.M. and E. Al., Extending the limits of
quantitative proteome profiling with data\_x0002\_independent
acquisition and application to acetaminophen-treated three dimensional
liver microtissues. Molecular \& Cellular Proteomics, 2015. 14(5): p.
1400-1410.

{[}23{]}. Han, D., et al., Signal Transduction Pathways Involved in
Drug-Induced Liver Injury. Handbook of Experimental pharmacology, 2009.
196(196): p. 267-310.

{[}24{]}. Nga T Nguyen, J.Y.A.J., Impaired protein adduct removal
following repeat administration of subtoxic doses of acetaminophen
enhances liver injury in fed mice. Arch Toxicol, 2021. 95(4): p.
1463-1473.

{[}25{]}. Hu, J.R.V.K., Low dose acetaminophen induces reversible
mitochondrial dysfunction associated with transient c-Jun N-terminal
kinase activation in mouse liver. Toxicological Sciences, 2016. 150(1):
p. 204--215.

{[}26{]}. Nguyen, N.U. and B.D. Stamper, Polyphenols reported to shift
APAP-induced changes in MAPK signaling and toxicity outcomes.
Chemico-Biological Interactions, 2017. 277: p. 129-136.

{[}27{]}. Gunawan, B.K., et al., c-Jun N-Terminal Kinase Plays a Major
Role in Murine Acetaminophen Hepatotoxicity. Gastroenterology, 2006.
131(1): p. 165-178.

{[}28{]}. Sharma M, G.V.J.A., Critical role for mixed-lineage kinase 3
in acetaminophen-induced hepatotoxicity. Mol Pharmacol, 2012. 82: p.
1001-1007.

{[}29{]}. Chenxi Shi, B.H.Y.Y., JNK Signaling Pathway Mediates
Acetaminophen-Induced Hepatotoxicity Accompanied by Changes of
Glutathione S-Transferase A1 Content and Expression. Front Pharmacol,
2019. 10: p. 1092.

{[}30{]}. Ramachandran, A. and H. Jaeschke, Acetaminophen
hepatotoxicity: A mitochondrial perspective. Advances in pharmacology
(1990), 2019. 85: p. 195-219.

{[}31{]}. Fan, X., et al., Pterostilbene Reduces Acetaminophen-Induced
Liver Injury by Activating the Nrf2 Antioxidative Defense System via the
AMPK/Akt/GSK3\selectlanguage{greek}β \selectlanguage{english}Pathway. Cellular Physiology and Biochemistry, 2018.
49(5): p. 1943-1958.

{[}32{]}. Du, K.F.A.J., Mitochondria-targeted antioxidant Mito-tempo
protects against acetaminophen hepatotoxicity. Archives of Toxicology,
2017. 91(2): p. 761-773.

{[}33{]}. Xie Y, R.A.B.D. and J.H. Farhood A, Inhibitor of apoptosis
signal-reg\_x0002\_ulating kinase 1 protects against
acetaminophen-induced liver injury. Toxicol Appl Pharmacol, 2015. 286:
p. 1-9.

{[}34{]}. Zhang J, M.R.L.K., The role of MAP2 kinases and p38 kinase in
acute murine liver injury models. Cell Death Dis, 2017. 8: p. e2903.

{[}35{]}. Shinohara, M.Y.M.D., Silencing glycogen synthase kinase-3beta
inhibits acetaminophen hepatotoxicity and attenuates JNK activation and
loss of glutamate cysteine ligase and myeloid cell leukemia sequence 1.
The Journal of Biological Chemistry, 2010. 285(11): p. 8244--8255.

.

{[}36{]}. Longjun Li, H.W.J.Z., SPHK1 deficiency protects mice from
acetaminophen-induced ER stress and mitochondrial permeability
transition. Cell Death Differ, 2020. 27(6): p. 1924-1937.

{[}37{]}. Du K, X.Y.M.M., Pathophysiological signifcance of c-Jun
N-terminal kinase in acetaminophen hepatotoxicity. Expert Opin Drug
Metab Toxicol, 2015. 11: p. 1769--1779.

{[}38{]}. Rong Zhang, R.A.L.B., Thioredoxin-2 inhibits
mitochondria-located ASK1-mediated apoptosis in a JNK-independent
manner. Circ Res, 2004. 94(11): p. 1483--1491.

{[}39{]}. Nakagawa H, M.S.H.Y. and K.M.I.H. Noguchi T, Deletion of
apoptosis signal-regulating kinase1 attenuates acetaminophen-induced
liver injury by inhibiting c-Jun N-terminal kinase activation.
Gastroenterology, 2008. 135(4): p. 1311--1321.

{[}40{]}. Sun, Y., et al., Liver-specific deficiency of unc-51 like
kinase 1 and 2 protects mice from acetaminophen-induced liver injury.
Hepatology, 2018. 67(6): p. 2397-2413.

{[}41{]}. Ni, H., et al., Activation of autophagy protects against
acetaminophen-induced hepatotoxicity. Hepatology, 2012. 55(1): p.
222-232.

{[}42{]}. Lee, S.K., et al., A phospholipase D2 inhibitor, CAY10594,
ameliorates acetaminophen-induced acute liver injury by regulating the
phosphorylated-GSK-3\selectlanguage{greek}β/\selectlanguage{english}JNK axis. Scientific Reports, 2019. 9(1).

{[}43{]}. Farkas Sarnyai, T.S.M.C., BGP-15 Protects Mitochondria in
Acute, Acetaminophen Overdose Induced Liver Injury. Pathol Oncol Res,
2020. 26(3): p. 1797-1803.

{[}44{]}. Win S, T.T.H.D., acetaminophen or tumor necrosis factor (TNF)
requires mitochondrial Sab protein expression in mice. J Biol Chem,
2011. 286: p. 35071--35078.

{[}45{]}. Sanda Win, T.A.T.R., c-Jun N\_x0002\_terminal kinase mediates
mouse liver injury through a novel Sab (SH3BP5)-dependent pathway
leading to inactivation of intra\_x0002\_mitochondrial Src. Hepatology,
2016. 63(06): p. 1987-2003.

{[}46{]}. Sandra Torres, A.B.N.I., Endoplasmic Reticulum Stress-Induced
Upregulation of STARD1 Promotes Acetaminophen-Induced Acute Liver
Failure. Gastroenterology, 2019. 157(2): p. 552-568.

{[}47{]}. Kon, K., et al., Mitochondrial permeability transition in
acetaminophen-induced necrosis and apoptosis of cultured mouse
hepatocytes. Hepatology, 2004. 40(5): p. 1170-1179.

{[}48{]}. Tsuruta F, S.J.M.Y. and M.N.G.Y. Yoshioka K, JNK promotes Bax
translocation to mitochondria through phosphorylation of 14-3-3
proteins. EMBO J, 2004. 23: p. 1889--1899.

{[}49{]}. Bong-Jo Kim, S.R., JNK- and p38 kinase-mediated
phosphorylation of Bax leads to its activation and mitochondrial
translocation and to apoptosis of human hepatoma HepG2 cells. J. Biol.
Chem., 2006. 281(30): p. 21256--21265.

{[}50{]}. Boelsterli UA, L.P., Mitochondrial abnormalities -- a link to
idiosyncratic drug hepato\_x0002\_toxicity? Toxicol Appl Pharmacol,
2007. 220(1): p. 92--107.

{[}51{]}. Bajt ML, F.A.L.J., Mitochondrial bax translocation accelerates
DNA fragmentation and cell necrosis in a murine model of acetaminophen
hepatotoxicity. J Pharmacol Exp Ther, 2008. 324: p. 8-14.

{[}52{]}. Bajt ML, F.A.L.J., Nuclear translocation of endonuclease G and
apoptosis-inducing factor during acetaminophen-induced liver cell
injury. Toxicol Sci, 2006. 94: p. 217--225.

{[}53{]}. He L, L.J., Regulated and unregulated mitochondrial
permeability transition pores: a new paradigm of pore structure and
function? FEBS Lett, 2002. 512: p. 1--7.

{[}54{]}. Nieminen AL, B.A.H.B., Mitochondrial permeability transition
in hepatocytes induced by t-BuOOH: NAD(P)H and reactive oxygen species.
Am J Physiol, 1997. 272: p. C1286 --1294.

{[}55{]}. Bajt ML, L.J.J.H., Role of mitochondrial Bax translocation in
acetaminophen-induced hepatic necrosis. Toxicol Sci, 2005: p. 84(Suppl
1):215.

{[}56{]}. Schroeter, H.B.C.S., c-Jun N-terminal kinase (JNK)-mediated
modulation of brain mitochondria function: new target proteins for JNK
signaling in mitochondrion-dependent apoptosis. Biochem. J, 2003. 372:
p. 359--369.

{[}57{]}. Kaplowitz, N., Biochemical and cellular mechanisms of toxic
liver injury. Semin. Liver Dis., 2002. 22(2): p. 137--144.

{[}58{]}. Xie, Y.M.M.R., Mechanisms of acetaminophen-induced cell death
in primary human hepatocytes. Toxicology and Applied Pharmacology, 2014.
279(3): p. 266--274.

{[}59{]}. Tournier, C.H.P.Y., Requirement of JNK for stress-induced
activation of the cytochromec-mediated death pathway. Science, 2000.
288: p. 870--874.

{[}60{]}. Sies H, S.V.K.L., Glutathione peroxidase protects against
peroxynitrite-mediated oxidations: a new function for selenoproteins as
peroxynitrite reductase. J Biol Chem, 1997. 272: p. 27812--27817.

{[}61{]}. K. Du, E.A., Resveratrol prevents protein nitration and
release of endonu\_x0002\_cleases from mitochondria during acetaminophen
hepatotoxicity. Food Chem.Toxicol., 2015. 81: p. 62-70.

{[}62{]}. Han, D.A.F.C., Voltage-dependent anion channels control the
release of the superoxide anion from mitochondria to cytosol. The
Journal of Biological Chemistry, 2003. 278(8): p. 5557--5563.

{[}63{]}. Saberi, B., et al., Protein kinase C (PKC) participates in
acetaminophen hepatotoxicity through c-jun-N-terminal kinase
(JNK)-dependent and -independent signaling pathways. Hepatology, 2014.
59(4): p. 1543-1554.

{[}64{]}. Del R\selectlanguage{ngerman}ío, S., et al., On the use of MapReduce for imbalanced
big data using Random Forest. Information Sciences, 2014. 285(0): p.
112-137.

{[}65{]}. Hiroki Aoki, P.M.K.J., Direct activation of mitochondrial
apoptosis machinery by c-Jun N-terminal kinase in adult cardiac
myocytes. J. Biol. Chem., 2002. 12: p. 10244--10250.

{[}66{]}. Kunimaro Furuta, Y.Y.S.O., Gab1 adaptor protein acts as a
gatekeeper to balance hepatocyte death and proliferation during
acetaminophen-induced liver injury in mice. Hepatology, 2016. 63(4): p.
1340-55.

{[}67{]}. Anup Ramachandran, et al., Receptor Interacting Protein Kinase
3 Is a Critical Early Mediator of Acetaminophen-induced Hepatocyte
Necrosis in Mice. HEPATOLOGY, 2013. 58: p. 2099-2108.

{[}68{]}. Lily Dara, Heather Johnson and Jo Suda, Receptor Interacting
Protein Kinase 1 Mediates Murine Acetaminophen Toxicity Independent of
the Necrosome and Not Through Necroptosis. Hepatology, 2015. 62(6): p.
1847-1857.

{[}69{]}. Zhang DW, S.J.L.J., RIP3, an energy metabolism regulator that
switches TNF-induced cell death from apoptosis to necrosis. Science,
2009. 325: p. 332-336.

{[}70{]}. Andrea Iorga, K.D.L.S., Interaction of RIPK1 and A20 modulates
MAPK signaling in murine acetaminophen toxicity. J Biol Chem, 2021. 296:
p. 100300.

{[}71{]}. Randi J Parks, S.M.K.M., Cyclophilin D-mediated regulation of
the permeability transition pore is altered in mice lacking the
mitochondrial calcium uniporter. Cardiovasc Res, 2019. 115(2): p.
385-394.

{[}72{]}. K. Itoh, T.C.S.T., An Nrf2/small Maf heterodimer mediates the
induction of phase II detoxifying enzyme genes through antioxidant
response elements. Biochemical and biophysical research communications,
1997. 236(2): p. 313-22.

{[}73{]}. J. R. Noh, Y.H.K.J., Sulforaphane protects against
acetaminophen-induced hepatotoxicity. Food and chemical toxicology : an
international journal published for the British Industrial Biological
Research Association 80, 2015: p. 193-200.

{[}74{]}. Chen, Y., et al., Carbon monoxide ameliorates
acetaminophen-induced liver injury by increasing hepatic HO-1 and Parkin
expression. The FASEB Journal, 2019. 33(12): p. 13905-13919.

{[}75{]}. Y. Chen, K.L.J.Z., JNK Phosphorylates The Neh6 Domain Of Nrf2
And Downregulates Cytoprotective Genes In Acetaminophen-Induced Liver
Injury. Hepatology, 2020.

{[}76{]}. Wang, L., et al., Farrerol Ameliorates APAP-induced
Hepatotoxicity via Activation of Nrf2 and Autophagy. International
Journal of Biological Sciences, 2019. 15(4): p. 788-799.

{[}77{]}. Yiping Chen, K.L.J.Z., c-Jun NH 2 -Terminal Protein Kinase
Phosphorylates the Nrf2-ECH Homology 6 Domain of Nuclear Factor
Erythroid 2-Related Factor 2 and Downregulates Cytoprotective Genes in
Acetaminophen-Induced Liver Injury in Mice. Hepatology, 2020. 71(5): p.
1787-1801.

{[}78{]}. Zhou, Z. and J. Qi, Exogenous activation of toll-like receptor
5 signaling mitigates acetaminophen-induced hepatotoxicity in mice.
Toxicol Lett, 2021. 324(15): p. 58-72.

{[}79{]}. Jaeschke, H.M.M.R., Current issues with acetaminophen
hepatotoxicity--a clinicallyrelevant model to test the efficacy of
natural products. Life Sci., 2011. 88: p. 737-745.

{[}80{]}. Hill, S.A.V.R., Mitochondrial stress signaling in longevity: a
new role formitochondrial functionin aging. Redox Biol., 2014. 2: p.
936--944.

{[}81{]}. Wang, H., et al., Double deletion of PINK1 and Parkin impairs
hepatic mitophagy and exacerbates acetaminophen-induced liver injury in
mice. Redox Biology, 2019. 22: p. 101148.

{[}82{]}. Dongshi Chen, H.N.L.W., p53 Up-regulated Modulator of
Apoptosis Induction Mediates Acetaminophen-Induced Necrosis and Liver
Injury in Mice. Hepatology, 2019. 65(2): p. 2164-2179.

{[}83{]}. De Smaela E, Z.F.P.S., Induction of gadd45\_x0004\_ by NF-kB
downregulates pro-apoptotic JNK signaling. Nature, 2001. 414: p.
308-312.

{[}84{]}. Eminel S, K.A.R.L., JNK2 translocates to the mitochondria and
mediates cytochrome c release in PC12 cells in response to
6-hydroxydopamine. J Biol Chem, 2004. 279: p. 55385--55392.

{[}85{]}. Meredith MJ, R.D., Status of the mitochondrial pool of
glutathione in the isolated hepatocyte. J Biol Chem, 1982. 257: p.

{[}86{]}. Gujral JS, K.T.F.A., Mode of cell death after acetaminophen
overdose in mice: apoptosis or oncotic necrosis? Toxicol Sci, 2002. 67:
p. 322--328.

{[}87{]}. Nagai H, M.K.F.G., Reduced glutathione de\_x0002\_pletion
causes necrosis and sensitization to tumor necrosis factor -induced
apoptosis in cultured mouse hepatocytes. HEPATOLOGY, 2002. 36: p. 55-64.

{[}88{]}. Bae, G.H., et al., Lysophosphatidic acid protects against
acetaminophen-induced acute liver injury. Experimental \& Molecular
Medicine, 2017. 49(12): p. e407-e407.

{[}89{]}. A. Chowdhury, J.L.R.Z., Mangiferin ameliorates
acetaminophen-induced hepatotoxicity through APAP-Cys and JNK
modulation. Biomedecine \& pharmacotherapie, 2019. 117: p. 109097.

{[}90{]}. M Faris K M Latinis, S.J.K., Stress-induced Fas ligand
expression in T cells is mediated through a MEK kinase 1-regulated
response element in the Fas ligand promoter. Mol. Cell. Biol., 1998. 18:
p. 5414--5424.

{[}91{]}. Lawson JA, F.A.H.R., The hepatic inflammatory response after
acetaminophen overdose: role of neutrophils. Toxicol Sci, 2000. 54: p.
509 --516.

{[}92{]}. Jaeschke H, G.G.C.A., Mechanisms of hepatotoxicity. Toxicol
Sci, 2002. 65: p. 166 --176.

{[}93{]}. Liu H, L.C.C.M., NF-kB inhibition sensitizes hepatocytes to
TNF-induced apoptosis through a sustained activation of JNK and c-Jun.
HEPATOLOGY, 2002. 35: p. 772-778.

{[}94{]}. Williams CD, B.M.F.A., Acetaminophen\_x0002\_induced hepatic
neutrophil accumulation and inflammatory liver injury in CD18-deficient
mice. Liver Int, 2010. 30: p. 1280-1292.

{[}95{]}. Jingyao Zhang, S.Z.J.B., Astaxanthin pretreatment attenuates
acetaminophen-induced liver injury in mice. Int Immunopharmacol, 2017.
45: p. 26-33.

{[}96{]}. James, L.P.K.R., Tumour necrosis factor receptor 1 and
hepatocyte regeneration in acetaminophen toxicity: a kinetic study of
proliferating cell nuclear antigen and cytokine expression. Basic Clin
Pharmacol Toxicol, 2005. 97: p. 8-14.

{[}97{]}. Spinnenhirn, V., J. Demgenski and T. Brunner, Death Receptor
Interactions With the Mitochondrial Cell Death Pathway During Immune
Cell-, Drug- and Toxin-Induced Liver Damage. Frontiers in Cell and
Developmental Biology, 2019. 7.

{[}98{]}. Kaufmann, T.J.P.J., Fatal hepatitis mediated by tumor necrosis
factor TNF\selectlanguage{greek}α \selectlanguage{english}requires caspase-8 and involves the BH3-only proteins bid
and bim. Immunity, 2009. 35: p. 56-66.

{[}99{]}. Schmich, K.S.R.C., Tumor necrosis factor \selectlanguage{greek}α \selectlanguage{english}sensitizes primary
murine hepatocytes to Fas/CD95-induced apoptosis in a Bim- and
Bid-dependent manner. Hepatology, 2010. 53: p. 282--292.

{[}100{]}. Badmann, A.K.A.K., Role of TRAIL and the pro-apoptotic Bcl-2
homolog Bim in acetaminophen-induced liver damage. Cell Death Dis, 2011.
2: p. e171.

{[}101{]}. Makoto Ishii, Y.S.K.T., Inhibition of c-Jun NH2-Terminal
Kinase Activity Improves Ischemia/Reperfusion Inhibition of c-Jun NH2
-Terminal Kinase. The Journal of Immunology, 2015.

{[}102{]}. Dharminder Chauhan, G.L.T.H., JNK-dependent release of
mitochondrial protein, Smac, during apoptosis in multiple myeloma (MM)
cells. J. Biol. Chem, 2003. 278(20): p. 17593--17596.

{[}103{]}. Yoshida, H.K.Y.Y., Apaf1 is required for mitochondrial
pathways of apoptosis and brain development. Cell, 1998. 94: p.
739--750.

{[}104{]}. Rajat Singh, M.J.C., Regulation of hepatocyte apoptosis by
oxidative stress. J. Gastroenterol. Hepatol., 2007. 22: p. Suppl.
1:S45--S48.

{[}105{]}. Yamamoto, K.I.H.A., BCL-2 is phosphorylated and inactivated
by an ASK1/Jun N-terminal protein kinase pathway normally activated at
G(2)/M. Mol. Cell. Biol, 1999. 19: p. 8469--8478.

{[}106{]}. Gross, A.M.J.M., BCL-2 family members and the mitochondria in
apoptosis. Genes Dev, 1999. 13: p. 1899-1911.

{[}107{]}. GANIATSAS, S., L. KWEE and Y. FUJIWARA, SEK1 deficiency
reveals mitogen-activated protein kinase cascade crossregulation and
leads to abnormal hepatogenesis. Proc Natl Acad Sci U S A, 1998. 95(12):
p. 6881--6886.

{[}108{]}. Liu, Z.G.H.H., Dissectionof TNF receptor 1 effector
functions: JNK activation is not linked to apoptosis while NF-kB
activation prevents cell death. Cell, 1996. 87: p. 565--576.

{[}109{]}. Francisco Javier Cubero, M.E.Z.J., Combined activities of
JNK1 and JNK2 in hepatocytes protect against toxic liver injury.
Gastroenterology, 2016.

{[}110{]}. McGill, M.R., et al., The mechanism underlying
acetaminophen-induced hepatotoxicity in humans and mice involves
mitochondrial damage and nuclear DNA fragmentation. The Journal of
clinical investigation, 2012. 122(4): p. 1574-1583.

{[}111{]}. Jaeschke H, C.C.B.M., Role of caspases in
acetaminophen\_x0002\_induced liver injury. Life Sci, 2006. 78(15): p.
1670--1676.

{[}112{]}. Yang, C., et al., Gasdermin D protects against noninfectious
liver injury by regulating apoptosis and necroptosis. Cell Death \&
Disease, 2019. 10(7).

{[}113{]}. Ming-Shiun Tsai, Y.W.Y.L. and G.L.J.K. Hsi-Kai Tsou,
Kaempferol protects against propacetamol-induced acute liver injury
through CYP2E1 inactivation, UGT1A1 activation, and attenuation of
oxidative stress, inflammation and apoptosis in mice. Toxicology
Letters, 2018.

{[}114{]}. Xiaoyong Li, J.L.Y.L., Hydrogen sulfide protects against
acetaminophen-induced acute liver injury by inhibiting apoptosis via the
JNK/MAPK signaling pathway. J Cell Biochem, 2018. 120(3): p. 4385-4397.

{[}115{]}. Zhou YD, H.J.L.W., 20(R)-ginsenoside Rg3, a rare saponin from
red ginseng, ameliorates acetaminophen-induced hepatotoxicity by
suppressing PI3K/AKT pathway-mediated inflammation and apoptosis. Int
Immunopharmacol, 2018. 59: p. 21--30.

{[}116{]}. Cao P, S.J.S.M., Angelica sinensis polysaccharide protects
against acetaminophen-induced acute liver injury and

cell death by suppressing oxidative stress and hepatic apoptosis in vivo
and in vitro. Int J Biol Macromol, 2018. 111: p. 1133--1139.

{[}117{]}. Uzi D, B.L.S.V., CHOP is a critical regulator of
acetaminophen-induced hepatotoxicity. J Hepatol, 2013. 59(03): p.
495--503.

{[}118{]}. Bajt ML, R.A.Y.H., Apoptosis-inducing factor modulates
mitochondrial oxidant stress in acetaminophen hepatotoxicity. Toxicol
Sci, 2011. 122(02): p. 598--605.

{[}119{]}. Kim JS, H.L.A.L., Mitochondrial permeability transition: a
common pathway to necrosis and apoptosis. Biochem Biophys Res Commun,
2003. 304: p. 463-- 470.

{[}120{]}. Crompton M, V.S.D.V., The mitochondrial permeability
transition pore. Biochem Soc Symp, 1999. 66: p. 167--179.

{[}121{]}. Badmann, A.L.S.K., TRAIL enhances paracetamol-induced liver
sinusoidal endothelial cell death in a Bim- and Bid-dependent manner.
Cell Death Dis, 2012. 3: p. e447.

{[}122{]}. G-Y Cheng, Q.J.A.D., CD31 induces inflammatory response by
promoting hepatic inflammatory response and cell apoptosis. Eur Rev Med
Pharmacol Sci, 2018. 22(21): p. 7543-7550.

{[}123{]}. Yan, T.W.H.Z., Glycyrrhizin Protects against
Acetaminophen-Induced Acute Liver Injury via Alleviating Tumor Necrosis
Factor alpha-Mediated Apoptosis. Drug Metab Dispos, 2016. 44: p.
720-731.

{[}124{]}. Possamai, L.A.M.M., Character and temporal evolution of
apoptosis in acetaminophen-induced acute liver failure. Crit Care Med,
2013. 41: p. 2543-2550.

{[}125{]}. Henderson CJ, W.C.K.N., Increased resistance to acetaminophen
hepatotoxicity in mice lacking glutathione S-transferase Pi. PNAS, 2000.
97: p. 12741-12745.

{[}126{]}. Jaeschke, H., A. Ramachandran and X. Chao, Emerging and
established modes of cell death during acetaminophen - induced liver
injury. Archives of Toxicology, 2019. 93: p. 3491-3502.

{[}127{]}. Junfeng An, F.M.C.H., ARC is a novel therapeutic approach
against acetaminophen\_x0002\_induced hepatocellular necrosis. J
Hepatol, 2013. 58: p. 297-305.

{[}128{]}. Liao, Y., et al., Oroxyloside ameliorates
acetaminophen-induced hepatotoxicity by inhibiting JNK related apoptosis
and necroptosis. Journal of Ethnopharmacology, 2020. 258: p. 112917.

{[}129{]}. Li, W.C.R.K., Isolation and culture of adult mouse
hepatocytes. Methods Mol. Biol., 2010. 633: p. 185--196.

{[}130{]}. Huo Y, Y.S.Y.M., Protective role of p53 in acetaminophen
hepatotoxicity. Free Radic Biol Med, 2017. 106: p. 111--117.

{[}131{]}. J. Y. Akakpo, A.R.L.D., Delayed Treatment With
4-Methylpyrazole Protects Against Acetaminophen Hepatotoxicity in Mice
by Inhibition of c-Jun n-Terminal Kinase. Toxicological sciences : an
official journal of the Society of Toxicology, 2019. 170(1): p. 57-68.

{[}132{]}. J Y Akakpo, A.R.S.E., 4-Methylpyrazole protects against
acetaminophen hepatotoxicity in mice and in primary human hepatocytes.
Hum Exp Toxicol, 2018. 37(12): p. 1310-1322.

{[}133{]}. Yong-Hoon Kim, J.N.J.H., Hepatocyte SHP deficiency protects
mice from acetaminophen-evoked liver injury in a JNK-signaling
regulation and GADD45\selectlanguage{greek}β-\selectlanguage{english}dependent manner. Arch Toxicol, 2018. 92(8): p.
2563-2572.

{[}134{]}. Y. H. Kim, J.H.H.K., Metformin ameliorates acetaminophen
hepatotoxicity via Gadd45beta\_x0002\_dependent regulation of JNK
signaling in mice. J Hepatol, 2015. 63(1): p. 75-82.

{[}135{]}. S. Papa, F.Z.Y.X., Gadd45\selectlanguage{greek}β \selectlanguage{english}promotes hepatocyte survival
during liver regeneration in mice by modulating JNK signaling. The
Journal of Biomedical Research, 2008. 118(5): p. 1911-1923.

{[}136{]}. V. L. Truong, E.A., Quercitrin from toona sinensis (juss.)
M.Roem. Attenuates acetaminophen-induced acute liver toxicity in HepG2
cells and mice through induction of antioxidant machinery and inhibition
of inflammation. Nutrients, 2016. 8(7).

{[}137{]}. Wenyan Xie, M.W.C.C., Hepatoprotective effect of
isoquercitrin against acetaminophen-induced liver injury. Life Sci,
2016. 152: p. 180-189.

{[}138{]}. Yuanyuan Li, J.X.D.L., Guavinoside B from Psidium guajava
alleviates acetaminophen-induced liver injury via regulating the Nrf2
and JNK signaling pathways. Food Funct, 2020. 11(9): p. 8297-8308.

{[}139{]}. Fu-Chao Liu, H.Y.A.C., Corilagin reduces
acetaminophen-induced hepatotoxicity through MAPK and NF- \selectlanguage{greek}κ \selectlanguage{english}B signaling
pathway in a mouse model. Am J Transl Res, 2020. 12(9): p. 5597-5607.

{[}140{]}. W Li, G.Y.Q.Z., TLR4 promotes liver inflammation by
activating the JNK pathway. Eur Rev Med Pharmacol Sci, 2019. 23(17): p.
7655-7662.

{[}141{]}. Yandong Zhang, Y.B.X.C., Protective effect of Chushizi
(Fructus Broussonetiae) on acetaminophen-induced rat hepatitis by
inhibiting the Toll-like receptor 3/c-Jun N-terminal
kinase/c-jun/c-fos/janus protein tyrosine kinase/activators of
transcription 3 pathway. J Tradit Chin Med, 2021. 40(6): p. 965-973.

{[}142{]}. Lung-Che Chen, L.H.M.Y., Alleviative effects from boswellic
acid on acetaminophen-induced hepatic injury - Corrected and republished
from: Biomedicine (Taipei). 2016 Jun; 6 (2): 9. doi:
10.7603/s40681-016-0009-1PMCID: PMC4864770. Biomedicine (Taipei), 2017.
7(2): p. 13.

{[}143{]}. Woolbright BL, J.H., Role of the inflammasome in
acet\_x0002\_aminophen-induced liver injury and acute liver failure. J
Hepatol, 2017. 66(04): p. 836--848.

{[}144{]}. Zuzana Papackova, M.H.H.D., Silymarin prevents
acetaminophen-induced hepatotoxicity in mice. PLoS One, 2018. 13(1): p.
e0191353.

{[}145{]}. Huifang Li, Y.L.J.L., Mannan-binding lectin attenuates
acetaminophen-induced hepatotoxicity by regulating CYP2E1 expression via
ROS-dependent JNK/SP1 pathway. Eur J Immunol, 2019. 49(4): p. 564-575.

{[}146{]}. Dietrich Conze, et al., c-Jun NH 2 -Terminal Kinase (JNK)1
and JNK2 Have Distinct Roles in CD8 T Cell Activation. journal of
experimental medicine, 2002. 195(7): p. 811--823.

{[}147{]}. Liu, Z.X.G.S., Innate immune system plays a critical role in
determining the progression and severity of acetaminophen
hepatotoxicity. Gastroenterology, 2004. 127: p. 1760--1774.

{[}148{]}. Antoniades, C.G.Q.A., Source and characterization of hepatic
macrophages in acetaminophen-induced acute liver failure in humans.
Hepatology, 2012. 56: p. 735--746.

{[}149{]}. Kiso, K.U.S.F., The role of Kupffer cells in carbon
tetrachloride intoxication in mice. Biol. Pharm. Bull., 2012. 35: p.
980--983.

{[}150{]}. Kaplowitz, N., Idiosyncratic drug hepatotoxicity. Nat.
Rev.Drug Discov, 2005. 4: p. 489--499.

{[}151{]}. Das, M., et al., The role of JNK in the development of
hepatocellular carcinoma. Genes \& development, 2011. 25(6): p. 634-645.

{[}152{]}. Haolu Wang, L.J.B.J., Imaging-based vascular-related
biomarkers for early detection of acetaminophen-induced liver injury.
Theranostics, 2020. 10(15): p. 6715-6727.

{[}153{]}. Bharat Bhushan, H.C.P.B., Dual Role of Epidermal Growth
Factor Receptor in Liver Injury and Regeneration after Acetaminophen
Overdose in Mice. Toxicol Sci, 2017. 155(2): p. 363-378.

{[}154{]}. Kluwe, J., et al., Modulation of Hepatic Fibrosis by
c-Jun-N-Terminal Kinase Inhibition. Gastroenterology, 2010. 138(1): p.
347-359.

{[}155{]}. Chowdhury Apu, et al., Current etiological comprehension and
therapeutic targets of

acetaminophen-induced hepatotoxicity. Pharmocological research, 2020.

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